Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), and penicillin–streptomycin were supplied by Gibco-BRL (Cergy Pontoise, France). Collagenase was obtained from Worthington Biochemical Corporation (Freehold, NJ, USA). Dispase, hyaluronidase, DNase I, diosgenin ([25R]-5α-spirosten-3β-ol), 4',6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and monoclonal antibody β-actin were purchased from Sigma (Saint Quentin Fallavier, France). 5B5 and JC/70A monoclonal antibodies and secondary polyclonal antibody conjugated with peroxidase were purchased from Dako (Trappes, France). RMO52 monoclonal antibody and fluorescein (DTAF)-conjugated goat anti-mouse antibody were purchased from Immunotech (Marseilles, France). COX-2 monoclonal antibody was supplied by Santa Cruz Biotechnology (TEBU; Le Perray en Yvelines, France). JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide) was supplied by Molecular Probes (Leiden, The Netherlands). CaspACE™ Assay System Fluorometric was supplied by Promega (Charbonnieres, France). Cell Death Detection ELISA^plus was supplied by Roche Diagnostics (Meylan, France). Celecoxib was obtained from Pharmacia (Skokie, IL, USA). PGE₂ and ELISA kits for PGE₂ were purchased from Cayman Chemical (SpiBio, Massy, France). Recombinant human IL-1β and Quantikine^® human IL-6 and IL-8 immunoassay kits were purchased from R&D Systems (Lille, France).

RA synoviocytes were isolated from fresh synovial biopsies obtained from six RA patients undergoing hip arthroplasty. All patients fulfilled the 1987 American Rheumatism Association criteria for RA 26. The mean age of the patients was 62.2 ± 4.6 years (range 55–68 years). The mean disease duration was 9.3 ± 2.2 years. At the time of surgery, the disease activity score (DAS 28) was greater than 3.2. These activities were approved by local institutional review boards, and all subjects gave written informed consent. Synovia were minced and digested with 1.5 mg/ml collagenase-dispase, 1 mg/ml hyaluronidase, and 0.15 mg/ml DNase I for 3–4 hours at 37°C as previously described 9. After centrifugation, cells were resuspended in DMEM supplemented with 10% FCS, 4.5 g/l D-glucose, 25 mM Hepes, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL) in a humidified atmosphere containing 5% (v/v) CO₂ at 37°C. After 48 hours, nonadherent cells were removed. Adherent cells (macrophage-like and FLS) were cultured in complete medium, and, at confluence, cells were trypsinized and only the FLS were passed. These cells were used between passages 4 and 8, when they morphologically resembled FLS after indirect immunofluorescence study (see Culture of human RA FLS). RA FLS were cultured 45–60 days before experimentation. This delay allowed the elimination of all possible interactions resulting from any preoperative treatment (with nonsteroidal anti-inflammatory drugs, analgesics, disease-modifying antirheumatic drugs, or steroids).

Between passages 4 and 8, RA FLS were trypsinized. Cell count and viability were determined and cells were plated in culture plates or flasks (Falcon, Oxnard, CA, USA). Viability, measured by trypan blue dye exclusion 27 at the start and the end of culture, was always greater than 95%. FLS (10⁵) from RA patients were used for indirect immunofluorescence study 28. The following monoclonal antibodies were used: 5B5 (anti-prolyl hydroxylase) for fibroblasts at 1/50 dilution (Dako, Burlingame, CA, USA), JC/70A (anti-CD31) for endothelial cells at 1/50 (Dako), and RMO52 (anti-CD14) for macrophages at 1/50 (Immunotech). The negative control was a mouse antibody of the same isotype (Immunotech). Incubations were performed at room temperature for 30 min. Binding of monoclonal antibodies was visualized using fluorescein (DTAF)-conjugated goat anti-mouse antibody (Immunotech) at 1/50 dilution.

For all experiments, RA FLS were allowed to adhere and grow for 48 hours in culture medium before exposure to diosgenin. A stock solution of 10^-2 M diosgenin was prepared in ethanol and diluted in culture medium to give a final concentration of 10–80 μM. The same amount of ethanol (<0.4%) was added to control cells. The culture medium was not changed during the entire study.

Cell proliferation was measured using the MTT assay. Cells (10³ cells/well) were plated in 96-well culture plates and grown for 48 hours before treatment with 10–80 μM diosgenin for 24–96 hours. MTT was carried out daily as previously described 29 and experiments were performed in sextuple assays.

For light microscopy, after 24–72 hours of treatment, RA FLS cultured cells were fixed in PBS (pH 7.4) containing 4% paraformaldehyde for 20 min at room temperature and washed in PBS for 15 min. Observations were made with phase-contrast microscopy.

Δψm was estimated using JC-1 (Molecular Probes). This is a fluorescent compound that exists as a monomer at low concentrations. At higher concentrations, it forms aggregates. Fluorescence of the JC-1 monomer is green, whereas that of the aggregate is red. Mitochondria with intact membrane potential concentrate JC-1 into aggregates, which fluoresce red, whereas de-energized mitochondria cannot concentrate it and fluoresce green 30.

Human RA FLS were grown for 48 hours before treatment with 40μM diosgenin for 24 hours. Control cells were grown in medium containing the same amount of ethanol as treated cells. Adherent cells were incubated in 1 ml of medium containing JC-1 (1 μg/ml) for 30 min at 37°C and pictures were taken with a Nikon microscope ECLIPSE E800 (Nikon Corporation, Champigny sur Marne, France). Moreover, human RA FLS were stained with DAPI (0.5 μg/ml) for 5 min at room temperature in the dark and the cells were examined by fluorescence microscopy.

After 40 μM diosgenin treatment for 24 or 48 hours, human RA FLS were homogenized in lysis buffer in accordance with the manufacturer's protocol (CaspACE™ Assay System Fluorometric, Promega). Fluorometric assays were conducted in white, opaque tissue-culture plates (Falcon, Becton Dickinson Labware, NJ, USA) and all measurements were carried out in triplicate. First, 100 μl of assay buffer (10 mM dithiothreitol, dimethyl sulfoxide, caspase buffer) (Promega) was added to each well. Peptide substrate for caspase-3 (Ac-DEVD–AMC [N-acetyl-Asp-Glu-Val-Asp–7-amino-4-methylcoumarin]) was added to each well to a final concentration of 2.5 mM. Caspase inhibitor (Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-aldehyde]) at 2.5 mM was also used just before the addition of the substrate. The supernatant of each cell lysate collected was added to each well to start the reaction. Background fluorescence was determined in wells containing assay buffer and substrate without cell lysate. Assay plates were incubated at 37°C for 1 hour for the measurement of caspase-3 activity. Fluorescence was measured with a microplate reader (Fluorolite 1000, Dynex Technologies, Chantilly, VA, USA) using 360 nm excitation and 460 nm emission filters. Raw data (relative units of fluorescence) corresponded to the concentrations of 7-amino-4-methylcoumarin released 31.

Human RA FLS were cultured in six-well culture plates (2 × 10⁵ cells/well). After diosgenin treatment (40 μM for 24 or 48 hours), apoptosis was quantified on pooled cells (floating and adherent) using the 'cell death' ELISA (Cell Death Detection ELISA^plus, Roche Diagnostics). Cytosol extracts were obtained in accordance with the manufacturer's protocol and apoptosis was measured as previously described 32. Other conditions are represented by cells pretreated for 4 hours at 37°C with IL-1β (1 ng/ml) or celecoxib (1 μM) and then 40 μM diosgenin was added in each flask for 24 or 48 hours in DMEM containing 10% (v/v) FCS in an atmosphere of 5% CO₂. We verified that under our experimental conditions 1 μM celecoxib was not proapoptotic. To examine the role of exogenously added PGE₂, we preincubated cells for 4 hours with COX-2 inhibitor (celecoxib 1 μM) in the absence or presence of PGE₂ (10 nM), followed by incubation with 40 μM diosgenin for an additional 24 hours.

Human RA FLS were cultured in 150-cm² tissue-culture flasks. After treatment with 40 μM diosgenin for 24 or 48 hours or with IL-1β (1 ng/ml) for 24 hours, adherent cells were trypsinized and pooled with the floating cell fraction. Western blot analysis was performed as previously described 32, using the primary monoclonal antibodies β-actin (mouse anti-human β-actin [1:5000], Sigma), COX-2 (mouse anti-human COX-2 [1:100], Santa Cruz Biotechnology), and secondary polyclonal antibody conjugated with peroxidase (Dako). Blots were visualized using enhanced chemiluminescence reagents (Amersham Biosciences, Orsay, France) and immediately exposed to x-ray film.

Human RA FLS were grown in 25-cm² tissue-culture flasks for 48 hours before treatment. After washing with PBS (pH 7.4), cells were pretreated for 4 hours at 37°C with IL-1β (1 ng/ml) or celecoxib (1 μM) and then 40 μM diosgenin was added in each flask for 24 or 48 hours in DMEM containing 10% (v/v) FCS in an atmosphere of 5% CO₂. Other conditions are represented by cells incubated with 40 μM diosgenin alone, IL-1β (1 ng/ml) alone, or celecoxib (1 μM) alone for 24 or 48 hours. The PGE₂ concentration in the medium was measured using an ELISA kit in accordance with the instructions of the manufacturer (Cayman Chemical) and was normalized with respect to the number of viable cells present in the particular culture at the time of sampling.

Human RA FLS were grown in 25-cm² tissue-culture flasks for 48 hours before treatment. After washing with PBS (pH 7.4), cells were pretreated for 4 hours at 37°C with IL-1β (1 ng/ml) and then 40 μM diosgenin was added to each flask for 24 or 48 hours in DMEM containing 10% (v/v) FCS in an atmosphere of 5% CO₂. Other conditions are represented by cells incubated with 40 μM diosgenin alone or IL-1β (1 ng/ml) alone for 24 or 48 hours. The cytokine concentration in the medium was measured using a Quantikine^® human IL-6 or IL-8 immunoassay kit in accordance with the instructions of the manufacturer (R&D Systems) and was normalized with respect to the number of viable cells present in the particular culture at the time of sampling.

The median and standard deviation (SD) were calculated using Excel (Microsoft Office, Version 98). Statistical analysis of differences was carried out by analysis of variance (ANOVA) using StatView Version 5.0 (SAS Institute Inc, Cary, NC, USA). A P-value of less than 0.05 (Fisher's protected-least-significant-difference test) was considered to indicate significance.

Cells were cultured in 10% FCS medium with or without 10–80 μM diosgenin for 24–96 hours and cell proliferation was evaluated by the MTT test. Under our experimental conditions, a dramatic decrease in proliferation was observed until 24 hours after diosgenin treatment (40 and 80 μM) (Fig. 1), especially at 24 hours for 40 μM diosgenin, when the percentage of inhibition was 76% (P < 0.05). As the percentage of inhibition did not strongly increase at 80 μM (79%; P < 0.05) (Fig. 1), we chose 40 μM for subsequent experiments.

Direct observation with phase-contrast microscopy demonstrated that human RA FLS treated with 40 μM diosgenin showed numerous morphological differences from control cells (Fig 2a). Cell shrinkage, cytoplasm condensation, and formation of cytoplasmic filaments appeared after 40 μM diosgenin treatment for 24, 48, and 72 hours (Fig. 2b,2c,2d respectively).

To ascertain potential mechanisms by which diosgenin inhibited the human RA FLS proliferation rate, we studied the effect of diosgenin on Δψm, because alterations in mitochondrial structure and function have been shown to play a crucial role in apoptosis.

Δψm was analyzed in adherent RA FLS after 24 hours of treatment with diosgenin using the potential-dependent, aggregate-forming lipophilic cation JC-1. Fluorescence, seen in Fig. 3, showed Δψm differences. We found that diosgenin induced a decrease of Δψm in RA FLS, shown by the incorporation of JC-1 monomers into the mitochondria (fluorescence in green, Fig. 3b), compared with cytosolic J-aggregate formation at high membrane potential in control cells (fluorescence in red, Fig. 3a).

Moreover, the morphology of treated human RA FLS was examined by fluorescence microscopy after DAPI staining. Diosgenin treatment of human RA FLS altered the extracellular and nuclear membrane permeability, as is shown by the DAPI nuclear localisation (Fig. 3b) in comparison with untreated cells (Fig. 3a).

It is well known that apoptosis is characterized by chromatin condensation and DNA fragmentation and is mediated by the cysteine protease family called caspases, such as caspase-3, which is the major executioner of apoptosis.

In our study, caspase-3 activity and DNA fragmentation were analyzed in human RA FLS treated or not with 40 μM diosgenin for 24 or 48 hours. Caspase-3 activity was significantly increased over time (2-fold at 24 hours and 2.6-fold at 48 hours in the diosgenin-treated cells versus controls; P < 0.05) (Fig. 4).

Quantitative determination of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) was performed with ELISA. Results showed that DNA fragmentation was enhanced 7-fold (P < 0.05) in treated cells at 24 hours and strongly induced at 48 hours (19-fold; P < 0.05) in comparison with controls (Table 1).

Numerous studies have shown that COX-2 expression prevents apoptosis in cancer cells, especially in colon cancer, in contrast to other cell types, for which the effects of COX-2 and related PGE₂ in the regulation of apoptosis could very well be cell-type-specific. Here we show that diosgenin induced overexpression of COX-2 over time (Fig. 5). This overexpression was correlated with COX-2 activity. Indeed, PGE₂ production was increased over time after diosgenin treatment: 2.3- and 4.7-fold (P < 0.05) at 24 or 48 hours, respectively, in comparison with controls (Fig. 6).

To further develop these results, it would be interesting to know if COX-2 was directly associated with diosgenin-induced human RA FLS apoptosis. In order to clarify this point, we used a specific inhibitor of COX-2 activity to verify DNA fragmentation after diosgenin treatment and examined the effect of exogenously added PGE₂ on diosgenin-induced synoviocyte death. We also studied whether COX-2 induction with IL-1β could have a synergistic effect with diosgenin on DNA fragmentation.

Our results showed that pretreatment with celecoxib before diosgenin treatment inhibited COX-2 activity over time: PGE₂ production was decreased by 73% and 95% (P < 0.05) at 24 and 48 hours, respectively, in comparison with diosgenin alone (Table 2). DNA fragmentation was also studied using the same conditions, and we found that pretreatment with celecoxib before diosgenin treatment reduced the production of mononucleosomes and oligonucleosomes by 47% and 46% at 24 and 48 hours, respectively; P < 0.05) in comparison with diosgenin alone (Fig. 7a). In contrast, exogenous PGE₂ did not have the same effect as endogenous PGE₂: we showed that the addition of 10 μM exogenous PGE₂ alone or in the presence of celecoxib before diosgenin treatment reduced DNA fragmentation in comparison with diosgenin alone (Fig. 7b).

Stimulation of RA FLS with IL-1β before diosgenin treatment dramatically enhanced COX-2 activity over time: we showed that PGE₂ synthesis increased 38- and 139-fold (P < 0.05) over 24 and 48 hours, respectively, in comparison with diosgenin alone (Table 2). The addition of IL-1β alone had no effect on human RA FLS apoptosis (Fig. 8). On the contrary, DNA fragmentation increased significantly after stimulation of the cells with IL-1β before diosgenin treatment (2.7- and 3.6-fold at 24 or 48 hours respectively; P < 0.05) in comparison with diosgenin alone (Fig. 8).

Human RA FLS are major producers of IL-6 and IL-8 in synovium and these proinflammatory cytokines participate in the pathogenesis of RA. Moreover, it is known that proinflammatory cytokine production can be activated during apoptosis of other cell types.

Our results showed that only IL-8 production (Fig. 9b) was significantly increased after 48 hours of diosgenin treatment (3.3-fold; P < 0.05) in comparison with control. IL-6 secretion was not modified over time with 40 μM diosgenin in comparison with controls (Fig. 9a). Moreover, we showed that diosgenin had a synergistic effect with IL-1β stimulation on the production of IL-8 by human RA FLS (Fig. 9b) but not of IL-6 (Fig. 9a). IL-1β-stimulated IL-8 production was increased 1.5- and 2.2-fold (P < 0.05) after diosgenin treatment for 24 and 48 hours, respectively, in comparison with IL-1β alone (Fig. 9b).